WO2009069132A2 - Nouveaux inhibiteurs de transcriptase inverse - Google Patents

Nouveaux inhibiteurs de transcriptase inverse Download PDF

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WO2009069132A2
WO2009069132A2 PCT/IL2008/001555 IL2008001555W WO2009069132A2 WO 2009069132 A2 WO2009069132 A2 WO 2009069132A2 IL 2008001555 W IL2008001555 W IL 2008001555W WO 2009069132 A2 WO2009069132 A2 WO 2009069132A2
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compound
group
reverse transcriptase
hiv
compounds
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PCT/IL2008/001555
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WO2009069132A3 (fr
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Amnon Hizi
Alon Herschhorn
Abraham Nudelman
Michal Weitman
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Ramot At Tel Aviv University Ltd.
Bar-Ilan University
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07C275/32—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by singly-bound oxygen atoms
    • C07C275/34—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by singly-bound oxygen atoms having nitrogen atoms of urea groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
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    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C335/18—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by singly-bound oxygen atoms
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    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C335/20—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
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    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
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    • C07C335/30—Isothioureas
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    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
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    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
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    • C07—ORGANIC CHEMISTRY
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    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
    • G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention in some embodiments thereof, relates to compounds capable of inhibiting an activity of reverse transcriptase (RT) and to methods utilizing same in the treatment of retroviral infections such as acquired immune deficiency syndrome (AIDS) caused by a human immunodeficiency virus (HIV).
  • RT reverse transcriptase
  • AIDS acquired immune deficiency syndrome
  • HIV human immunodeficiency virus
  • Retroviruses are small, single-stranded positive-sense RNA viruses.
  • a retroviral particle comprises two identical single-stranded positive sense RNA molecules. Their genome contains, among other things, the sequence encoding the enzyme reverse transcriptase (RT). Many molecules of reverse transcriptase are found in close association with the genomic RNA in the mature viral particles. Upon entering a cell, this reverse transcriptase produces a double-stranded DNA copy of the viral genome, which is then inserted into the chromatin of a host cell. Once inserted, the viral sequence is called a pro virus. Retroviral integration is directly dependent upon viral proteins. Linear viral DNA termini (the LTRs) are the immediate precursors to the integrated proviral DNA. There is a characteristic duplication of short stretches of the host's DNA at the site of integration.
  • LTRs Linear viral DNA termini
  • RT copies the viral (+) single stranded genomic RNA into double stranded DNA.
  • This process is catalyzed solely by RT and depends on two fundamental activities: the DNA polymerase, which copies both RNA and DNA into DNA, and the ribonuclease H (RNase H) that concomitantly cleaves the viral RNA strand in the RNA/DNA heteroduplex.
  • RNase H ribonuclease H
  • the resulting double-stranded DNA is transported into the nucleus as part of a pre-integration complex and is subsequently incorporated into the DNA of the cell by the viral enzyme integrase.
  • HIV-I human immunodeficiency virus type-1
  • NRTIs nucleoside/nucleotide RT inhibitors
  • NRTIs non-nucleoside RT inhibitors
  • the NNRTIs include a variety of non-competitive inhibitors that bind specifically to a hydrophobic pocket in proximity to the DNA polymerase active site of the enzyme; most of them are highly specific against HIV-I RT with minimal effects on the closely-related HIV-2 RT. Both classes of inhibitors are currently used in therapy against HIV-I, as part of the highly active anti-retro viral therapy (HAART), which simultaneously targets the RT, the viral protease and most recently the entry step of the virus.
  • HAART highly active anti-retro viral therapy
  • HIV-I drugs are dominated by either nucleoside reverse transcriptase inhibitors or peptidomimetic protease inhibitors.
  • a triple-drug therapy regimen is often used to treat AIDS patients.
  • This "cocktail" includes a combination of a protease inhibitor called indinavirTM with two nucleoside reverse- transcriptase inhibitors, AZTTM and 3TCTM.
  • the triple drug therapy results in a decrease in measured levels of virus in both blood and lymphatic tissues.
  • the cocktail therapy is largely restricted by the emergence of resistant viral strains, high costs and long duration of the treatment.
  • NNRTIs Non-nucleoside reverse transcriptase inhibitors
  • nevirapine nevirapine
  • benzoxazinone efavirenz
  • bis(heteroaryl) piperazine derivatives delavirdine
  • Etravirine Etravirine
  • the major drawback to the development and application of NNRTIs is the propensity for rapid emergence of drug resistant strains, both in tissue cell culture and in treated individuals, particularly those subject to monotherapy.
  • NNRTIs A significant obstacle for the use of NNRTIs is their very high specificity that reduces their efficacy against mutated variants of the virus.
  • the protein targets for therapy especially the RT and protease, are quite flexible and can tolerate mutations and still remain active, resistance develops rapidly during treatment even when a combination of drugs is used.
  • Several mutant strains of HIV have been characterized, and resistance to known therapeutic agents is due to mutations in the RT gene.
  • Some of the most commonly observed mutants clinically are: the Y181C mutant, in which a tyrosine (Y), at codon 181, has been mutated to a cysteine (C) residue, and K103N where the lysine (K) at position 103 has been replaced by asparagine (N).
  • mutants which emerge with increasing frequency during treatment with known antivirals, include the single mutants V106A, G190A, Y188C, and P236L; and the double mutants K103N/Y181C, K103N/P225H, K103N/V108I, and K103N/L100I.
  • Virtual screening is a technique, in which each member of a large available chemical database is docked into the active site of an enzyme of interest. The compounds are then ranked according to their potential molecular interactions with the enzyme. Eventually, the top-scoring compounds can be obtained and tested for their capacity to inhibit in vitro the activity of the enzyme. Exemplary methodologies for virtual screening potential RT inhibitors are described in Herschhorn et al. [J Med. Chem. 2007 50, 2370-2384 and Biochemistry 2008, 47, 490-502].
  • Novel inhibitors against HIV-I RT are usually identified by screening a very large number of compounds against the recombinant RT enzyme and testing the compounds for their ability to protect susceptible cells from a productive HIV-I infection.
  • the latter method also requires an additional step of identifying, among the active inhibitors, those compounds that are specifically directed against HIV-I RT (since compounds not active against HIV-I RT may suppress viral infectivity by other mechanisms).
  • screenings and the subsequent optimization of the inhibitors by systematic chemical modifications are highly time-and resource-consuming.
  • De novo design of new molecules is done by first identifying functional groups that bind the active site of the target protein and then linking them into the final inhibitor molecules. The molecules are then synthesized and tested for their enzymatic inhibitory effects.
  • the present inventors have designed and successfully practiced a novel methodology for identifying compounds that inhibit reverse transcriptase.
  • two different crystalline forms of wild type HIV-I RT were used, and a chemical library of 46,000 compounds was screened for molecules that could bind independently to each of the tested crystalline forms.
  • Those molecules that interacted with both RT crystalline forms were subsequently tested in vitro for inhibiting an activity of HIV-I RT, and a plurality of molecules were identified as potent RT inhibitors.
  • additional, novel compounds were designed, prepared and tested, and were also found as potent inhibitors of HIV- 1 RT activity.
  • Z is selected from the group consisting of a substituted or non-substituted aryl and heteroaryl, wherein when substituted, the substituent is selected from the group consisting of halide, alkoxy, hydroxy, thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R 4 ;
  • Z is a substituted or non-substituted aryl having formula II:
  • Ra-Re are each independently selected from the group consisting of halide, alkoxy, hydroxy, thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R 4 , wherein at least one of the Ra-Rd is selected from the group consisting of halide, alkoxy, alkyl, carboxylate, cyano, nitro, haloalkyl and amine.
  • Ra is selected from the group consisting of alkoxy, alkyl and halide.
  • the alkoxy is methoxy.
  • Rb is hydrogen.
  • Rc is selected from the group consisting of hydrogen, halide and nitro.
  • Rd is selected from the group consisting of hydrogen, alkyl, halide, alkoxy and haloalkyl.
  • Rd is alkoxy.
  • W is S or O.
  • each of R 5 and R 6 is hydrogen.
  • the hydrocarbon chain has 2-4 carbon atoms. In some embodiments of the invention, R is a hydrocarbon chain having 2 or 3 carbon atoms.
  • R 9 is a hydrocarbon chain having 3 or 4 carbon atoms.
  • Z has the general formula II, wherein Ra is methoxy, Rb is hydrogen, Rc is chloro, Rd is methoxy and Re is hydrogen.
  • a pharmaceutical composition comprising the compound having Formula Ia as described herein and a pharmaceutically acceptable carrier.
  • the composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a viral infection caused by a retrovirus.
  • a process of synthesizing the compound having Formula IA as described herein comprising: reacting a compound having the formula Z-R 1 with a compound having the formula A-R 2 , wherein R 2 is amine and R 1 is selected such that upon reacting with the amine, the X is formed.
  • a method of inhibiting an activity of a reverse transcriptase comprising contacting the reverse transcriptase with a compound selected from the group consisting of:
  • Z is selected from the group consisting of a substituted or non-substituted aryl and heteroaryl, wherein when substituted, the substituent is selected from the group consisting of halide, alkoxy, hydroxy, thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R 4 ;
  • R and R are each independently a linear or branched, saturated or unsaturated substituted or non-substituted hydrocarbon chain having 1-10 carbon atoms in its backbone, which when substituted, the substituent is selected from the group consisting of alkyl and cycloalkyl;
  • R 4 , R 5 , R 6 , R 10 , R 1 1 , R 12 and R 13 are each independently hydrogen or alkyl; Cii) a compound having the general Formula III:
  • B 1 and B 2 are each independently selected from the group consisting of a substituted or unsubstituted heteroaryl and heteroalicyclic, which, when substituted, the substituent is selected from the group consisting of halide, alkoxy, hydroxy thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R ,
  • the compound has the general Formula I.
  • Z is a substituted or non- substituted aryl having formula II:
  • Ra-Re are each independently selected from the group consisting of halide, alkoxy, hydroxy, thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R 4 , provided that at least one of the Ra-Rd is selected from the group consisting of halide, alkoxy, alkyl, carboxylate, cyano, nitro, haloalkyl and amine.
  • Ra is selected from the group consisting of alkoxy, alkyl and halide. According to some embodiments of the invention, Ra is alkoxy.
  • Rb is hydrogen
  • Rc is selected from the group consisting of hydrogen, halide and nitro.
  • Rd is selected from the group consisting of hydrogen, alkyl, halide, alkoxy and haloalkyl.
  • Rd is alkoxy
  • W is S or O.
  • each of R 5 and R 6 is hydrogen.
  • the hydrocarbon chain has 2-4 carbon atoms.
  • R 8 is a hydrocarbon chain having 2 or 3 carbon atoms.
  • R 9 is a hydrocarbon chain having 3 or 4 carbon atoms.
  • Z has the general formula II, wherein Ra is methoxy, Rb is hydrogen, Rc is chloro, Rd is methoxy and Re is hydrogen.
  • the compound has the general formula
  • the reverse transcriptase is wild type HIV- 1 reverse transcriptase or a mutant thereof and at least one heteroatom in the B 1 and B 2 is capable of interacting with Glul38 of the p51 subunit in the HIV-I reverse transcriptase and/or Tyr318 in the p66 subunit in the HIV-I reverse transcriptase.
  • the compound is Compound 3.
  • the contacting is affected in vitro.
  • the contacting is affected in vivo.
  • the reverse transcriptase is selected from the group consisting of a wild type reverse transcriptase and a mutant thereof.
  • the reverse transcriptase is an
  • HIV-I reverse transcriptase or a mutant thereof.
  • the reverse transcriptase is an
  • HIV-2 reverse transcriptase or a mutant thereof.
  • the compound is characterized by a specific inhibition of the reverse transcriptase, as determined by the percent of residual activity of a cellular DNA polymerase in the presence of the compound divided by the percent of residual activity of the reverse transcriptase in the presence of the compound (i.e. the selectivity index).
  • the reverse transcriptase is wild type HIV-I -reverse transcriptase and the DNA polymerase is Klenow fragment of
  • the activity of the reverse transcriptase is selected from the group consisting of RT-associated DNA-dependent
  • RNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase activity.
  • a pharmaceutical composition comprising, as an active ingredient, a compound as described herein and a pharmaceutically acceptable carrier, the composition being packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a viral infection caused by a retrovirus.
  • a use of a compound as described herein in the preparation of a medicament for treating a viral infection caused by a retrovirus is provided.
  • a compound as described herein being identified for use in the treatment of a viral infection caused by a retrovirus.
  • a method of treating a viral infection caused by a retrovirus comprising administering to a subject in need thereof a therapeutically effective amount of a compound as described herein.
  • the retrovirus is HIV-I.
  • the retrovirus is selected from the group consisting of HIV-I and HIV-2.
  • any of the pharmaceutical compositions as described herein further comprises at least one agent capable of treating a viral infection caused by a retrovirus.
  • the compound in any of the uses described herein is utilized in combination with at least one agent capable of treating a viral infection caused by a retrovirus.
  • the method as described herein comprises administering to the subject a therapeutically effective amount of at least one agent capable of treating a viral infection caused by a retrovirus.
  • the at least one agent is selected from the group consisting of a protease inhibitor, a reverse transcriptase inhibitor, an integrase inhibitor and any combination thereof.
  • the at least one agent is selected from the group consisting of 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxyinosine (ddl), 2',3'-dideoxycytidine (ddC), d4T, 3TC, dipyridodiazepinone (nevirapine), benzoxazinone (efavirenz) and bis(heteroaryl) piperazine derivatives (delavirdine), abacavir and any combination thereof.
  • AZT 3'-azido-3'-deoxythymidine
  • ddl 2',3'- dideoxyinosine
  • ddC 2',3'-dideoxycytidine
  • d4T 3TC
  • dipyridodiazepinone nevirapine
  • benzoxazinone efavirenz
  • bis(heteroaryl) piperazine derivatives delavirdine
  • a method of identifying a candidate compound for inhibiting an activity of a reverse transcriptase comprising computationally identifying a compound which is capable of specifically binding to a three-dimensional structure of an active site cavity of at least two crystalline forms of a reverse transcriptase, thereby identifying the candidate compound for inhibiting an activity of the type reverse transcriptase.
  • the reverse transcriptase is selected from the group consisting of a HIV-I reverse transcriptase and a HIV-2 reverse transcriptase.
  • the reverse transcriptase is selected from the group consisting of a wild type reverse transcriptase and a mutant thereof.
  • the reverse trascriptase is a wild type HIV-I reverse transcriptase.
  • the reverse transcriptase is a wild type HIV-I reverse transcriptase and the crystalline forma of the HIV-I reverse transcriptase have the pdb entry codes Ifk9 and ldtq.
  • the computationally identifying comprises:
  • the docking process is performed by fragmenting each compound and fitting the conformation of each fragment into the protomol to yield a spatial structure that maximizes molecular similarity to the protomol.
  • the identifying is by computational means.
  • the method further comprises biologically assaying the candidate compound for its activity in inhibiting a catalytic activity of the reverse transcriptase. According to some embodiments of the invention, the method further comprises biologically assaying the candidate compound for its activity in treating a viral infection caused by a retrovirus.
  • the method is being for identifying candidate compounds for treating a viral infection caused by a retrovirus.
  • all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
  • methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
  • Implementation of the method and/or system of embodiments of the invention can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and/or system of the invention, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system.
  • hardware for performing selected tasks according to embodiments of the invention could be implemented as a chip or a circuit.
  • selected tasks according to embodiments of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system.
  • one or more tasks according to exemplary embodiments of method and/or system as described herein are performed by a data processor, such as a computing platform for executing a plurality of instructions.
  • the data processor includes a volatile memory for storing instructions and/or data and/or a non-volatile storage, for example, a magnetic hard-disk and/or removable media, for storing instructions and/or data.
  • a network connection is provided as well.
  • a display and/or a user input device such as a keyboard or mouse are optionally provided as well.
  • a protein or “at least one protein” may include a plurality of proteins, including mixtures thereof.
  • the term “comprising” means that other steps and ingredients that do not affect the final result can be added. This term encompasses the terms “consisting of and “consisting essentially of.
  • the term “method” or “process” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • FIGs. IA-D present the chemical structure and inhibitory effect of Compound 20, an exemplary reverse transcriptase inhibitor according to some embodiments of the present invention, and the inhibitory effect of Compound 3, another exemplary reverse transcriptase inhibitor according to some embodiments of the present invention.
  • FIG. IA presents images demonstrating the inhibition effect of various concentrations of Compound 20 on HIV-I infectivity of B lymphocytes observed through a fluorescence microscope (left column) and plots demonstrating the HIV-I infectivity measured quantitatively by flow cytometry (right column).
  • FIG. IB presents the 2D chemical structure of Compound 20.
  • FIG. ID presents the toxicity and therapeutic index of Compounds 3 and 20 on B lymphocytes, wherein CC 5O is the concentration of inhibitor at which the cell viability was reduced by 50 % compared to that of the inhibitor-free control.
  • TI is the therapeutic index (CC 5O /IC 5O ).
  • FIG. 2 presents plots demonstrating the inhibition of wild type HIV-I RT by Compound 3 (left column) and Compound 20 (middle column), exemplary reverse transcriptase inhibitors according to some embodiments of the present invention, compared with the known NNRTI, nevirapine (right column).
  • Wild type RT, incubated with the tested compounds was assayed for DNA-dependent DNA polymerase activity (DDDP; top raw), RNA-dependent DNA polymerase activity (RDDP; middle raw) and RNase H activity (bottom raw).
  • the RDDP, DDDP, and RNase H activities of wild- type HIV-I RT were assayed in the presence of increasing concentrations of the tested compounds.
  • FIGs. 3 A-B present plots demonstrating the inhibition of Yl 81C mutant of HIV-
  • FIG. 3A a bar graph demonstrating the inhibition of HIV-2 RT (FIG. 3B) by Compounds 3 and 20, exemplary reverse transcriptase inhibitors according to some embodiments of the present invention, and the known NNRTI, nevirapine.
  • Y181C mutant of HIV-I RT incubated with Compound 3 (FIG. 3 A; left column), Compound 20 (FIG. 3A; middle column) and nevirapine (FIG. 3A; right column) was assayed for DNA-dependent DNA polymerase activity (DDDP).
  • the dose response curves for each inhibitor were fitted to a four-parameter logistic equation and the IC 50 parameters as well as standard errors, sigmoidity and correlation coefficients are reported.
  • a bar graph comparing the inhibitory effect of Compound 3, 20 and nevirapine, each at a concentration of 140 ⁇ M, on HIV-2 RT DNA-dependent polymerase activity (DDDP) is also shown (FIG. 3B).
  • FIG. 4 presents comparative plots showing of the maximal velocity of HIV-I RT DNA-dependent DNA polymerase activity as a function of enzyme concentration.
  • the DNA-dependent DNA polymerase activity was monitored by assessing the poly(rA) n « oligo(dT) I2-18 -dependent incorporation of [ 3 H]dTTP into nascent DNA in the absence (circles) or presence of 616 nM of Compound 20 (triangle).
  • RT activity was assayed for 15 minutes at 37 0 C and the extent of the [ 3 HJdTTP incorporation was determined as described in the Examples section.
  • the curves were fitted by linear regression analysis that resulted in high correlation coefficients (r2) of 0.995 in the absence of an inhibitor, and 0.998 in the presence of an inhibitor, indicating a strong linear relationship between V ma ⁇ values and enzyme concentrations.
  • FIGs. 5A-D present kinetic analysis of the inhibition of HIV-I RT-associated DNA-dependent DNA polymerase activity by Compound 20, an exemplary reverse transcriptase inhibitor according to some embodiments of the present invention, with respect to either the dTTP substrate (FIGs. 5A-B) or the rA»dT template (FIGs. 5C-D).
  • FIGs. 5 A and C present a double-reciprocal plot of the initial velocity of the RNA- dependent DNA polymerase activity of HIV-I RT as a function of dTTP substrate concentration or rA » dT template concentration, respectively.
  • the velocity was measured with increasing concentrations of dTTP or the rA # dT template tested in the absence (filled square) or presence of 0.25 ⁇ M (filled circle), 0.5 ⁇ M (star), 1 ⁇ M (filled diamond) or 2 ⁇ M (filled triangle) of Compound 20FIGs. 5B and 5D present a Replot (Dixon plot) of the reciprocal maximal velocity (calculated from FIGs. 5A and 5C, respectively) versus various concentrations of Compound 20.
  • the kinetic constants K m and Ki were calculated using linear regression analysis.
  • FIGs. 6A-B present a computer generated structural model of the docking of Compounds 3 and 20, exemplary reverse transcriptase inhibitors according to some embodiments of the present invention, into the NNRTI binding site of wild-type HIV-I RT.
  • FIG. 4 A presents the spatial structure of wild-type HIV-I RT with its defined subdomains (left, each subdomain in the p66 subunit is depicted in a different color while the p51 is positioned behind the p66) and the location of the hydrophobic pocket along with the three aspartic acids that form the catalytic site of the DNA polymerase domain (right).
  • FIG. 4 A presents the spatial structure of wild-type HIV-I RT with its defined subdomains (left, each subdomain in the p66 subunit is depicted in a different color while the p51 is positioned behind the p66) and the location of the hydrophobic pocket along with the three aspartic acids that form the catalytic site of the DNA polymerase domain (right).
  • 6B presents the docking of Compounds 3 (left) and 20 (right) into the HIV-I RT structure as found in PDB entry ldtq using Surflex.
  • the top-scoring conformation of each molecule is shown.
  • Amino acids in the pocket are specified in three-letter codes and different colors; Glul38 is part of the p51 HIV-I RT subunit, and all the other amino acids are part of the p66 subunit.
  • the two compounds are depicted in CPK (Corey, Pauling, and Kultin) colors (i.e. nitrogen blue, oxygen red sulfur yellow and carbon gray) and are displayed as a stick model. Suggested hydrogen bonds are displayed as dashed lines. All structures were displayed with discovery studio visualizer 1.6 (Accelrys Software Inc.).
  • the present invention in some embodiments thereof, relates to compounds capable of inhibiting an activity of reverse transcriptase (RT) and to uses thereof in the treatment of retroviral infections such as acquired immune deficiency syndrome (AIDS) caused by a human immunodeficiency virus (HIV).
  • RT reverse transcriptase
  • HIV human immunodeficiency virus
  • HIV-I human immunodeficiency virus type-1
  • RT inhibitors Numerous RT inhibitors have been identified so far as agents for treating AIDS. These are divided into nucleoside/nucleotide analogs RT inhibitors (NRTIs) and the non-nucleoside/nucleotide RT inhibitors (NNRTIs).
  • NRTIs nucleoside/nucleotide analogs RT inhibitors
  • NRTIs non-nucleoside/nucleotide RT inhibitors
  • the NNRTIs include a variety of non-competitive inhibitors that bind specifically to a hydrophobic pocket in proximity to the DNA polymerase active site of the enzyme; most of them are highly specific against HIV-I RT with minimal effects on the closely-related HIV-2 RT.
  • RT inhibitors As discussed hereinabove, the use of currently marketed RT inhibitors in the treatment of retroviral infections such as AIDS is restricted by the emergence of drug resistant mutants of RT.
  • This method is based on (i) in silco virtual screening of a chemical library for candidate compounds which dock onto the NNRTI binding pocket of the RT; and (ii) in vitro testing these candidate compounds for the selective inhibition of the RT-associated DNA polymerase activity, as further described in detail hereinbelow, so as to identify candidate compounds that are indeed characterized as capable of inhibiting this RT activity.
  • a chemical library is a collection of stored chemicals which can be used ultimately in high-throughput screening or industrial manufacture.
  • a chemical library thus consists a series of stored chemicals, whereby for each chemical, an associated information such as its chemical structure, purity, and physicochemical characteristics , is also stored. Utilizing the above-described methodology, the present inventors have identified candidate compounds and have further tested their HIV-I RT inhibition activity.
  • a method of identifying a candidate compound for inhibiting an activity of a reverse transcriptase is effected by computationally-identifying a compound, which is capable of specifically- binding to a three-dimensional structure of an active site cavity of at least two crystalline forms of a reverse transcriptase.
  • the phrase "candidate compound” describes a compound that can potentially be utilized for an intended use, herein for inhibiting an activity of RT and/or for treating an infection caused by a retrovirus.
  • a candidate compound can act, for example, as a competitive inhibitor of RT activity, a non-competitive inhibitor of RT activity, a compound which interferes with RT binding to nucleosides, etc.
  • the phrase "competitive inhibitor”, as used in the context of the present embodiments, describes a compound which directly binds to the same active site as a natural substrate of the enzyme RT.
  • a competitive inhibitor typically binds the active site via reversible interactions, as in the case of a natural substrate, and thus prevents an interaction between at least some of the enzyme molecules with the natural substrate.
  • non-competitive inhibitor describes a compound that interacts with the enzyme, herein RT, at a site other than the enzyme's active site, such that this interaction affects its interaction with its natural substrate via, for example, a conformational or chemical change in the enzyme's structure .
  • An "active site cavity” describes one or more amino acid(s) within the enzyme's sequence that an interaction therewith affects the interaction of the enzyme with its natural substrate, in a competitive or non-competitive manner, as defined hereinabove.
  • the active site cavity is a hydrophobic pocket in proximity to the DNA polymerase active site of the RT.
  • Such an active site cavity is also referred to herein interchangeably as a hydrophobic binding site within the catalytic domain of the reverse transcriptase.
  • the RT inhibitors described herein are non-competitive inhibitors. It is shown, for example, that an exemplary compound which was uncovered via the screening method described herein, exhibited potent noncompetitive inhibition of RT activity (see, for example, Example 3 in the Examples section that follows). As used herein, the phrase "reverse transcriptase" encompasses a wild type RT and any mutant thereof.
  • the RT is HIV-I RT.
  • the method according to this aspect of the invention is effected by computationally identifying a compound which is capable of specifically binding to a three-dimensional structure of an active site cavity, as described herein, of a wild type of HIV-I reverse transcriptase.
  • a screening process which utilizes, in the docking, more than one crystalline form of the RT permits a more stringent screen that can lead to more reliable results, since it assures that subtle differences between the various RT structures will not affect the overall efficacy of the docking process.
  • the wild type reverse transcriptase is a HIV-I reverse transcriptase and the crystalline forms of this HIV-I reverse transcriptase have the pdb entry codes Ifk9 and ldtq.
  • the computational identification described herein is affected by:
  • the identification described in (e) above is also effected by computational means.
  • the active site can be represented by a protomol.
  • a protomol is an idealized representation of a ligand that makes every potential interaction with the selected active site and is an object-oriented component based framework for molecular dynamics simulations.
  • the active site is a hydrophobic pocket in proximity to the DNA polymerase binding site of the RT.
  • each compound is energy-minimized to a single, optimized conformation and a docking process of the compounds onto the active site is thereafter initiated, using the generated Protomol.
  • the docking process is performed by fragmenting each compound and fitting the conformation of each fragment into the protomol, so as to yield a spatial structure that maximizes molecular similarity to the protomol.
  • three-dimensional structure describes the orderly geometric spatial arrangement of atoms within the protein-of- interest (i.e., tertiary structure of the protein) and is defined by its atomic coordinates.
  • obtaining the set of atomic coordinates which defines the three dimensional structure of the active site cavity of an enzyme (e.g., HIV-I RT)
  • NMR nuclear magnetic resonance
  • X-ray crystallography is preferred for obtaining the secondary and tertiary structure information, which requires detailed information about the arrangement of atoms within a protein.
  • a three dimensional structure of a protein-of-interest can be constructed using computer-based protein modeling techniques.
  • the three dimensional structure of a protein is solved by finding target sequences that are most compatible with profiles representing the structural environments of the residues in known three-dimensional protein structures (See, e.g., U.S. Pat. No. 5,436,850).
  • the known three-dimensional structures of proteins in a given family are superimposed in order to define the structurally conserved regions of that protein family.
  • This protein modeling technique also uses a known three- dimensional structure of a homologous protein to approximate the structure of a polypeptide of interest (See, e.g., U.S. Pat. Nos. 5,557,535; 5,884,230; and 5,873,052).
  • computer-readable medium refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. Selection and use of appropriate storage media is well within the capabilities of one of ordinary skill in the art.
  • recorded refers to a process of storing information on computer readable medium.
  • the coordinate data used to define the structures of the active site cavities of the RTs is retrieved from a published database (Research Collaboratory for Structural Bioinformatics site) as a complex with a known RT inhibitor (an NNRTI). Further computational manipulations can be performed so as to obtain the coordinate data of an unbound enzyme and/or to obtain the coordinate data of mutants that cannot be retrieved from an available database.
  • structure models of the present embodiments are preferably generated by a computing platform, which generates a graphic output of the models via a display generating device such as screen or printer.
  • the computing platform generates graphic representations of atomic structure models via a processing unit which processes structure coordinate data stored in a retrievable format in the data storage device.
  • Suitable software applications may be used by the processing unit to process structure coordinate data so as to provide a graphic output of three-dimensional structure models generated therewith via display. Criteria employed by software programs used in in silico screening processes to qualify the binding of screened chemical structures with binding pockets include gap space, hydrogen bonding, electrostatic interactions, Van der Walls forces, hydrophilicity/hydrophobicity, etc.
  • Contact area between compounds may be directly calculated from the coordinates of the compounds in docked conformation using the MS program (Connolly ML., 1983. Science 221, 709-713).
  • Docking may be followed by energy minimization with standard molecular mechanics force fields or dynamics with programs such as CFF 1.02 force field.
  • minimization of energy means achieving an atomic geometry of a chemical structure via systematic alteration such that any further minor perturbation of the atomic geometry would cause the total energy of the system, as measured by a molecular mechanics force-field, to increase. Minimization and molecular mechanics force fields are well understood in computational chemistry.
  • candidate compounds namely, candidate RT inhibitors
  • these compounds are further tested for their in vitro RT inhibition activity, so as to identify those compounds which may serve as potent RT inhibitors.
  • the candidate compounds detected in the virtual screening process were further tested in vitro for their HIV-I RT inhibition activity (see, Tables 2, 3 and 4).
  • RNase activity was evaluated.
  • the inhibition activity of the compounds was quantified by determining the level of inhibition of the residual activity of RT by the compounds.
  • the residual activity was calculated by dividing the activity, detected in the presence of a specific tested compound, by the initial activity with no compound present, as detailed in the Examples section that follows.
  • the compounds described herein exhibit a high level of selectivity and specificity towards HIV-I RT, as compared to other unrelated cellular DNA polymerases (see, Tables 2 and 4).
  • This selectivity and specificity (designated as the selectivity index) was calculated by dividing the residual activity of an unrelated cellular DNA polymerases (such as
  • the compound is characterized by a specific inhibition of a reverse transcriptase, as determined by the percent of residual activity of a cellular DNA polymerase in the presence of the compound divided by the percent of residual activity of the reverse transcriptase in the presence of said compound
  • selectivity index (also referred to herein as a selectivity index).
  • the selectivity index is measured using a HIV-I -reverse transcriptase and a DNA polymerase being Klenow fragment of E. coli DNA polymerase I (KF).
  • the method described herein further comprises biologically assaying the compound for its activity in inhibiting a catalytic activity of a reverse transcriptase, whereby compounds identified as inhibitors of RT, as described hereinabove, are further identified as capable of treating a viral infection caused by a retrovirus.
  • the screening method described herein can therefore be utilized for identifying compounds that can be utilized for treating a viral infection caused by a retrovirus.
  • the retrovirus is HIV (e.g., HIV-I or HIV-2).
  • HIV e.g., HIV-I or HIV-2.
  • HIV-I RT inhibition activity have been uncovered.
  • Z is selected from the group consisting of a substituted or non-substituted aryl and heteroaryl, wherein when substituted, the substituent is selected from the group consisting of halide, alkoxy, hydroxy, thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, solfonyl, sulfynyl, carboxylate, cyano, nitro, amide, and OCO 2 R 4 ;
  • A is R 8 - Y- R 9 , wherein Y is selected from the group consisting of-O-, -N-, -S-, -
  • R and R are each independently a linear or branched, saturated or unsaturated substituted or non-substituted hydrocarbon chain having 1-10 carbon atoms in its backbone, which when substituted, the substituent is selected from the group consisting of alkyl and cycloalkyl;
  • R 4 , R 5 , R 6 , R 10 , R 11 , R 12 and R 13 are each independently hydrogen or alkyl; CU) a compound having the general Formula III:
  • B 1 and B 2 are each independently selected from the group consisting of a substituted or unsubstituted heteroaryl and heteroalicyclic, which, when substituted, the substituent is selected from the group consisting of halide, alkoxy, hydroxy thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R 15 ;
  • amine as used herein, describes a -NR'R" group, wherein R' and R" are each independently hydrogen, alkyl, cycloalkyl, or hydroxy, as these terms are defined hereinbelow, unless otherwise indicated.
  • alkyl describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., "1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted.
  • the substituent can be, for example, alkyl, cycloalkyl, aryl, heteroaryl, halide, trihalomethyl, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, carboxylate, carbamyl, thiocarbamyl, sulfinyl, sulfonyl, amide and amine as defined herein.
  • cycloalkyl describes an all-carbon monocyclic or fused ring ⁇ i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system.
  • the cycloalkyl group may be substituted or unsubstituted.
  • the substituent can be, for example, alkyl, cycloalkyl, aryl, heteroaryl, halide, trihalomethyl, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, carboxylate, carbamyl, thiocarbamyl, sulfinyl, sulfonyl, amide and amine as defined herein.
  • aryl describes an all-carbon monocyclic or fused-ring polycyclic ⁇ i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted.
  • the substituent can be, for example, alkyl, cycloalkyl, aryl, heteroaryl, halide, trihalomethyl, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, carboxylate, carbamyl, thiocarbamyl, sulfinyl, sulfonyl, amide and amine as defined herein.
  • heteroaryl describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • the heteroaryl group may be substituted or unsubstituted.
  • the substituent can be, for example, alkyl, cycloalkyl, aryl, heteroaryl, halide, trihalomethyl, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, carboxylate, carbamyl, thiocarbamyl, sulfinyl, sulfonyl, amide and amine as defined herein.
  • a “heteroalicyclic” group describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
  • the heteroalicyclic may be substituted or unsubstituted.
  • the substituent can be, for example, alkyl, cycloalkyl, aryl, heteroaryl, halide, trihalomethyl, hydroxyl, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, carbonyl, thiocarbonyl, carboxylate, carbamyl, thiocarbamyl, sulfinyl, sulfonyl, amide and amine, as defined herein.
  • halide which is also referred to herein as "halo" describes fluorine, chlorine, bromine or iodine.
  • haloalkyl describes an alkyl group as defined hereinabove, further substituted by one or more halide(s).
  • An example is a trihaloalkyl, such as trifluoromethyl.
  • alkoxy describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
  • aryloxy describes both an -O-aryl and an -O-heteroaryl group, as defined herein.
  • hydroxyl describes a -OH group.
  • thiohydroxy which is also referred to herein as “thiol”, describes a -
  • thioalkoxy describes both a -S-alkyl group, and a -S-cycloalkyl group, as defined herein.
  • thioaryloxy describes both an -S-aryl and an -S-heteroaryl group, as defined herein.
  • cyano which is also referred to herein as nitrile, describes a -C ⁇ N group.
  • nitro describes an -NO 2 group.
  • hydrocarbon describes an organic moiety that includes, as its basic skeleton, a chain of carbon atoms, substituted mainly by hydrogen atoms.
  • the hydrocarbon can be saturated or non-saturated, be comprised of aliphatic, alicyclic or aromatic moieties, and can optionally be substituted by one or more substituents (other than hydrogen).
  • the compounds utilized in the context of the invention have the general formula III.
  • An exemplary compound which belongs to this category is compound 3 (see, Table 2 hereinbelow).
  • Compound 3 was found to be a potent HIV-RT inhibitor (see, Table 2 and Figures ID and Figure 2). It has further been shown that Compound 3 fits into the NNRTI hydrophobic binding site in HIV-I RT (See, Figure 6). Specifically, Molecular modeling studies have revealed that Compound 3 forms two important hydrogen bonds with Glul38 of the p51 subunit in the HIV-I reverse transcriptase and with Tyr318 in the p66 subunit in the HIV-I reverse transcriptase. Tyr318 is highly conserved in HIV-I RT and interacts within a 4 A contact distance with most NNRTIs that have been shown to bind the hydrophobic pocket.
  • At least one heteroatom in the B 1 and B 2 moieties in Formula III is capable of interacting with Glul38 of the p51 subunit in said HIV-I reverse transcriptase and/or with Tyr318 in the p66 subunit in said HIV-I reverse transcriptase.
  • compounds having general Formula HI are characterized as having a heteroaryl or heteroalicyclic moiety, in which a heteroatom thereof is capable of interacting with the above-described Glul38, and as further having an additional heteroaryl or heteroalicyclic moiety, in which a heteroatom thereof is capable of interacting with the above-described Tyr318.
  • the heteroaryl or heteroalicyclic moieties, and the D moiety linking therebetween are therefore selected such that both these interactions can occur.
  • heteroaryl or heteroalicyclic moieties, and the D moiety linking therebetween are selected such that the length, structure and flexibility of those portions of these moieties that link one heteroatom in Bl to one heteroatom in B2 allow at least one interaction between a heteroatom and the above-described Glul38 and another heteroatom and the above- described Tyr318.
  • R 16 is preferably hydrogen.
  • Bl is benzo[c][l,2,5]thiadiazole
  • B2 is lH-pyrazole.
  • the lH-pyrazole is substituted by a halide, for example, bromo.
  • a compound having general Formula III is Compound 3.
  • the compounds utilized in the context of the invention are selected from the group consisting of Compounds 1, 4, 5, 6, 8 and 13 (see, chemical structures in Tables 2 and 3).
  • compounds having Formula I have general Formula I.
  • compounds having Formula I have a thiourea moiety having attached to one nitrogen atom thereof an aryl moiety and to the other nitrogen atom thereof an aliphatic moiety interrupted by a heteroatom (see, Tables 2, 4 and 5). It has been suggested that the position and nature of the substituent on the aryl moiety has an effect on the potency of these compounds as RT inhibitors.
  • Z in formula I is a substituted or non- substituted aryl having Formula II:
  • Ra-Re are each independently selected from the group consisting of halide, alkoxy, hydroxy, thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R 4 , whereas at least one of the Ra-Rd is selected from the group consisting of halide, alkoxy, alkyl, carboxylate, cyano, nitro, haloalkyl and amine.
  • the halide is chlorine, fluorine or bromine. In some embodiments the halide is chlorine. In some embodiments, the alkoxy is methoxy or ethoxy, preferably methoxy.
  • the alkyl is a methyl, propyl or tert-butyl.
  • the haloalkyl is CF 3 .
  • Ra is selected from the group consisting of alkoxy, alkyl and halide.
  • exemplary compounds wherein Ra is an alkyl, such as methyl include Compounds 9, 25, 26 and 35 (see, Tables 2 and 4).
  • Compounds 9 and 35 exhibited potent HIV-I RT inhibition activity with a selectivity and specificity to HIV-I RT as compared to an unrelated cellular DNA polymerase (i.e. high selectivity index; see Table 2 and 4).
  • Compound 9 was further tested and was able to inhibit the transfection of cells by the HIV-I virions (see, Table 2).
  • Exemplary compounds wherein Ra is halide include Compounds 22, 30, 34 and 37 (see, Table 4).
  • Compound 22, in which Ra is chloride exhibited potent HIV-I RT inhibition activity with a high selectivity index and was also shown to inhibit the transfection of cells by the HIV-I virions (see, Table 4).
  • Ra is alkoxy. In some embodiments the alkoxy is methoxy.
  • An Exemplary compound in which Ra is ethoxy and which exhibited potent HIV-I RT inhibition activity is Compound 27. Exemplary compounds in which Ra is methoxy and which exhibited potent HIV-I RT inhibition activity include Compounds
  • Rb in Formula II is hydrogen.
  • Rc is selected from the group consisting of hydrogen, halide and nitro.
  • Exemplary compounds wherein Rc is hydrogen include Compounds 7, 22, 25, 27, 28, 29, 32, 33, 34 and 36 (see, Tables 2 and 4).
  • Compounds 7, 22, 27, 28 exhibited potent HIV-I RT inhibition activity with a high selectivity index and inhibited the transfection of cells by the HIV-I virions (see, Tables 2 and 4).
  • Exemplary compounds wherein Rc is halide include Compounds 2, 9, 19, 20, 21, 24, 30, 37, 38 (see, Tables 2 and 4) and Compounds 46, 49, 54, 62, 63, 67 and 70 (see, Table 5).
  • Compounds 2, 9, 20, 21 exhibited potent HIV-I RT inhibition activity together with a high selectivity index (see, Tables 2 and 4).
  • Compound 20 was especially selective toward HIV-I RT with a selectivity index of 7.4.
  • Compound 2, 9 and 20 were also able to inhibit the transfection of cells by the HIV-I RT virions (see, Tables 2 and 4 and Figure 1).
  • An exemplary compound wherein Rc is nitro and which exhibited potent HIV-I RT inhibition activity is Compound 44 (see, Table 5). Without being bound to any particular theory, it has been suggested that an electron withdrawing group at position Rc may have an effect on the inhibition activity of a compound having general Formula I.
  • Rd is selected from the group consisting of hydrogen, alkyl, halide, alkoxy and haloalkyl.
  • Exemplary compounds wherein Rd is methyl include Compounds 19, 22, 28 and 35 (see, Table 4).
  • Compounds 22, 28 and 35 exhibited potent HIV-I RT inhibition activity with a high selectivity index and HIV-I virions transfection inhibition (see, Table 4).
  • An Exemplary compound wherein Rd is chloride and which exhibited potent HIV-I RT inhibition activity is Compound 39.
  • Exemplary compounds wherein Rd is hydrogen include Compounds 9, 21, 23, 25, 26, 27, 29, 30, 31, 34, 37 and 40 (see, Tables 2 and 4).
  • Compounds 9, 21, 23, and 24 exhibited potent HIV-I RT inhibition activity with a high selectivity index. Compound 27 also exhibited HIV-I virions transfection inhibition.
  • An exemplary compound wherein Rd is haloalkyl and which exhibited potent HIV-I RT inhibition activity, high selectivity index and HIV-I virions transfection inhibition is Compound 2.
  • Exemplary compounds wherein Rd is methoxy and which exhibited potent HIV-I RT inhibition activity include Compounds 7, 20, 32 (see, Tables 2 and 4) and Compounds 44, 46, 49, 54, 59, 62, 68, 69 and 70 (see, Table 5). Without being bound to any particular theory, it has been suggested that the presence of a methoxy group at position Rd is essential for the inhibition activity of a compound having general Formula I.
  • W is S or O.
  • W CHNO 2 .
  • R 5 and R 6 are each hydrogen.
  • Y in formula I can be -O-, -N-, -S-, -CR 1 R 11 -,
  • Y is -O-.
  • Exemplary compounds in which Y is O and exhibited potent HIV-I RT inhibition include Compounds 7, 9 and 20-40 (see, Tables 2 and 4) and Compounds 44 and 46 (see, Table 5). As discussed hereinabove, Compounds 7, 9, 20, 22, 27, 28, and 39 exhibited inhibition of transfection of cells by
  • HIV-I virions see, Tables 2 and 4 and Figure 1).
  • Y is -S-.
  • An Exemplary compound in which Y is S and which exhibited potent HIV-I RT inhibition is Compound 54 (see, Table 5).
  • R 8 and R 9 in formula I are each independently a linear, saturated, non-substituted hydrocarbon chain having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms in its backbone.
  • the hydrocarbon chain has 2, 3 or 4 carbon atoms.
  • R 8 is a hydrocarbon chain having 2 or 3 carbon atoms.
  • R 9 is a hydrocarbon chain having 3 or 4 carbon atoms.
  • Exemplary compounds wherein each of R 8 and R 9 is a hydrocarbon chain having 3 carbon atoms and which exhibited potent HIV-RT activity include Compounds 20-40 (see, Table 4) and Compounds 44, 46, 54, 59, 67, 69 and 70 (see, Table 5).
  • An Exemplary compound wherein R 8 and R 9 are hydrocarbon chains having 2 and 4 carbon atoms, respectively, and which exhibited potent HIV-RT activity is Compound 62 (see, Table 5).
  • An Exemplary compound wherein R 8 and R 9 are hydrocarbon chains having 3 and 4 carbon atoms, respectively, and which exhibited potent HIV-RT activity is Compound 68 (see, Table 5).
  • An Exemplary compound wherein R and R are hydrocarbon chains having 2 and 3 carbon atoms, respectively, and which exhibited potent HIV-RT activity is Compound 63 (see, Table 5).
  • the total length of the aliphatic chain substituting the moiety X is Formula I, namely, the total number of carbon atoms in the hydrocarbon chains of R 8 and R 9 has an effect of the RT inhibition activity exhibited by compounds having Formula I.
  • the above- descried total length should be at least of 6 carbon atoms and can range, for example, from 6 to 10 carbon atoms, preferably from 6 to 8 carbon atoms.
  • the total number of carbon atoms in R 8 and R 9 together is higher than 6. In some embodiments the total number of atoms in R 8 , Y and R together is higher than 6. In some embodiments the total number is 7. In some embodiments the total number is 8.
  • Exemplary compounds which exhibited potent HIV-I RT inhibition activity and wherein the total number of atoms in R 8 , Y and R 9 together is higher than 6 include Compounds 20-40 (see, Table 4), Compounds 63, 54, 46, and 44 (having a total number of atoms being 7), Compounds 59, 62, 67, 69 and 70 (having a total number of atoms being 8) and Compound 68 (having a total number of atoms being 9) (see, Table 5).
  • Z has the general formula II, wherein Ra is methoxy, Rb is hydrogen, Rc is chloro, Rd is methoxy and Re is hydrogen.
  • W is O or S
  • each of R 5 and R 6 is hydrogen.
  • Exemplary compounds which belong to this category and exhibited potent HIV-I RT inhibition activity include Compound 20, and Compounds 46, 54, 59, 62, 63, 67, 68, 68 and 70.
  • Y and Ra-Re are as described hereinabove, and m and i are each independently an integer from 0-5, preferably from 0-2.
  • novel compounds which can be collectively represented by general Formula Ia: Z-X-A Formula Ia wherein:
  • Z is selected from the group consisting of a substituted or non-substituted aryl and heteroaryl, wherein when substituted, the substituent is selected from the group consisting of halide, alkoxy, hydroxy, thioalkoxy, thioalkyl, alkyl, haloalkyl, amine, sulfonamide, sulfinyl, sulfonyl, carboxylate, cyano, nitro, amide, and OCO 2 R 4 ;
  • R and R are each independently a linear or branched, saturated or unsaturated substituted or non-substituted hydrocarbon chain having 1-10 carbon atoms in its backbone, which when substituted, the substituent is selected from the group consisting of alkyl and cycloalkyl;
  • each of the variables in Formula Ia are as described hereinabove for Formula I. It is noted that while the novel compounds described herein share some structural features of Compound 20, there compounds include various modifications to the chemical structure of Compound 20. These include, for example, modifications of the aliphatic side chain of the thiourea moiety; modification of the thiourea moiety; and/or modification of the substituent of the aryl ring.
  • present embodiments further encompass any pharmaceutically acceptable salt, prodrug, solvate, hydrate and, if present, purified enantiomers of each of the compounds described herein.
  • pharmaceutically acceptable salt refers to a charged species of the parent compound and its counter ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound.
  • prodrug refers to an agent, which is converted into the active compound (the active parent drug) in vivo.
  • Prodrugs are typically useful for facilitating the administration of the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions. Prodrugs are also often used to achieve a sustained release of the active compound in vivo.
  • solvate refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the compound described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute.
  • Suitable solvents include, for example, ethanol, acetic acid and the like.
  • hydrate refers to a solvate, as defined hereinabove, where the solvent is water.
  • R 1 is isothiocyanate, which, upon reaction with amine, forms a thiourea moiety.
  • R 1 is isocyanate, which, upon reaction with amine, forms a urea moiety.
  • the compounds described herein were selected and/or designed capable of inhibiting an activity of a reverse transcriptase.
  • a method of inhibiting an activity of a reverse transcriptase which is effected by contacting the reverse transcriptase with any of the compounds described herein.
  • the reverse transcriptase can be a HIV-I RT and/or HIV-2 RT.
  • At least Compound 20 is capable of inhibiting an activity of both HIV-I RT and HIV-2 RT.
  • the contacting is effected in vitro. According to some embodiments the contacting is effected in vivo. According to some embodiments, the activity of the reverse transcriptase is RT- associated DNA-dependent DNA polymerase activity and/or RNA-dependent DNA polymerase activity.
  • DNA-dependent DNA polymerase activity describes the ability of a reverse transcriptase to use DNA as a template for transcription of DNA. This term is also referred to herein as "DNA polymerase activity”.
  • RNA-dependent DNA polymerase activity describes the ability of a reverse transcriptase to use RNA as a template for transcription of DNA. This term is also referred to herein as "RNA polymerase activity”.
  • the reverse transcriptase can be a wild type RT or a mutant thereof.
  • the compounds described herein can inhibit an activity of both a wild type HIV-I RT and of resistant mutants thereof.
  • the exemplary Compounds 3 and 20 inhibited the RNA-dependent DNA polymerase activity of the well known drug resistant mutant HIV-I RT Yl 81C (see, Figure 3A).
  • the phrase "wild type reverse transcriptase" describes an enzyme produced by an un-mutated cell.
  • mutant with respect to wild type RT describes an enzyme having an amino acid sequence which differs from that of the wild-type enzyme due to the genetic mutation of the virus that harbors this enzyme.
  • genetic mutation describes genetic alteration in the genome of a cell or a virus which in turn alters the amino acid sequence of the enzyme produced thereby.
  • the compounds described herein are capable of inhibiting a catalytic activity of a reverse transcriptase such as HIV-I reverse transcriptase.
  • the reverse transcriptase is an essential enzyme in the retroviral life cycle. Therefore, according to an aspect of some embodiments of the present invention there is provided a method of treating or preventing a viral infection caused by a retrovirus (also referred to herein as a retroviral infection). This method is effected by administering to a subject in need thereof a therapeutically effective amount of any of the compounds described herein, including a combination thereof.
  • the terms "treat,” “treating,” and “treatment”, as used herein, encompass alleviating or abrogating a disorder; or one or more of the symptoms associated with the disorder; or alleviating or eradicating the cause(s) of the disorder itself.
  • prevent refers to a method of delaying or precluding the onset of a disorder; and/or its attendant symptoms, barring a subject from acquiring a disorder or reducing a subject's risk of acquiring a disorder.
  • therapeutically effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder being treated.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • patient are used interchangeably herein in reference to a human subject.
  • virus describes a group of microbes which, with few exceptions, are capable of passing through fine filters that retain most bacteria, and are incapable of growth or reproduction in media apart from living cells. See, e.g., Stedman's Medical
  • retrovirus describes a class of viruses of vertebrate animals in which the genetic material is RNA, instead of DNA. Such viruses are accompanied by a polymerase enzyme known as "reverse transcriptase", which catalyzes transcription of viral single-stranded RNA into double-stranded DNA. The resultant DNA may remain in a dormant state in an infected cell for an indeterminate period of time, or become incorporated into the cells genome and actively cause the formation of new virions.
  • viral infection or “retroviral infection” describes any disease caused by retroviruses, particularly retroviruses pathogenic to subjects such as humans.
  • treating viral infection describes a compound's ability to inhibit viral infection of cells, via, for example, cell-cell fusion or free virus infection.
  • infections may involve membrane fusion, as occurs in the case of enveloped viruses, or some other fusion event involving a viral structure and a cellular structure (e.g., such as the fusion of a viral pilus and bacterial membrane during bacterial conjugation).
  • the reverse transcriptase can be derived from any retrovirus, including, for example, Moloney Murine Leukemia Virus (M-MLV), Rous Sarcoma Virus (RSV), Avian Myeloblastosis Virus (AMV), Mouse
  • M-MLV Moloney Murine Leukemia Virus
  • RSV Rous Sarcoma Virus
  • AMV Avian Myeloblastosis Virus
  • MMTV Tumor Virus
  • BLV Bovine Leukemia virus
  • the retrovirus is HIV-I.
  • AIDS related complex followed by AIDS.
  • Affected individuals exhibit severe immunosuppression which makes them highly susceptible to debilitating and ultimately fatal opportunistic infections.
  • Replication of HIV by a host cell requires integration of the viral genome into the host cell's DNA. Since HIV is a retrovirus, the HIV replication cycle requires transcription of the viral RNA genome into DNA by the HIV-RT.
  • the reverse transcriptase is a HIV-RT or a mutant thereof. Accordingly, the compounds described herein are particularly useful in the treatment (or prophylaxis) of HIV infections such as AIDS and associated conditions, such as AIDS related complex (ARC), Kaposi's sarcoma, and AIDS dementia.
  • HIV infections such as AIDS and associated conditions, such as AIDS related complex (ARC), Kaposi's sarcoma, and AIDS dementia.
  • AIDS related complex ARC
  • Kaposi's sarcoma Kaposi's sarcoma
  • AIDS dementia AIDS related complex
  • AIDS Acquired immunodeficiency syndrome
  • HAV human immunodeficiency virus
  • This condition progressively reduces the effectiveness of the immune system and leaves individuals susceptible to opportunistic infections and tumors.
  • HIV is transmitted through direct contact of a mucous membrane or the bloodstream with a bodily fluid containing HIV, such as blood and blood fractions, semen, vaginal fluid, preseminal fluid, and breast milk.
  • the symptoms of AIDS are primarily the result of conditions that do not normally develop in individuals with healthy immune systems. Most of these conditions are infections caused by bacteria, viruses, fungi and parasites that are normally controlled by the elements of the immune system that HIV damages.
  • Subjects with AIDS also have an increased risk of developing various cancers such as Kaposi's sarcoma, cervical cancer and cancers of the immune system known as lymphomas.
  • AIDS related complex is a condition in which antibody tests are positive for HIV. Patients with ARC show the mild symptoms of HIV infection, which include enlarged lymph nodes, fatigue, night sweats, weight loss, and diarrhea.
  • treatment of HIV infection refers to improvement in at least one clinical parameter associated with the HIV infection as compared to non-treated control and notably to improve in the viral load count and increase in CD4+ bearing cells.
  • the improvement may be actual reduction in the viral load, but may also be slowing down in the rate of increase of the viral load, slowing down of the physical deterioration, and side effects associated with AIDS.
  • HIV-2 is another strain of HIV retrovirus having the ability to infect human cells. HIV-2 retrovirus is found primarily in West Africa and is similar to HIV-I but appears less destructive to the immune system and has a slower disease progression. Most NNRTIs are highly specific against HIV- 1 RT with minimal effects on the closely-related HIV-2 RT. As exemplified in the example section which follows, the compounds described herein exhibited anti-HIV-2 activity with a profound inhibition of the DNA-dependent DNA polymerase activity of the enzyme as compared to the well known NNRTI nevirapine (see Figure 3B).
  • the reverse transcriptase is an HIV-2 reverse transcriptase or a mutant thereof.
  • the compounds of the present embodiments may be employed in combination with other therapeutic agents for the treatment of the above infections or conditions.
  • Combination therapies according to embodiments of the present invention comprise the administration of at least one compound as described herein and at least one other pharmaceutically active ingredient.
  • the compound(s) described herein and the other pharmaceutically active agents may be administered simultaneously in either the same or different pharmaceutical compositions or sequentially in any order.
  • the amounts of the compound(s) as described herein and the other pharmaceutically active agent(s) and the relative timings of administration are selected in order to achieve the desired combined therapeutic effect.
  • the compounds described herein are administered to a subject together with an additional agent (e.g., a pharmaceutically active agent), as a combination therapy as described hereinabove.
  • an additional agent e.g., a pharmaceutically active agent
  • the additional agent is capable of treating the viral infection from which the subject is suffering.
  • Preferred combination therapies include simultaneous or sequential treatment with any of the compounds described herein and one or more of the following:
  • reverse transcriptase inhibitors such as abacavir, adefovir, didanosine, lamivudine, stavudine, zalcitabine and zidovudine;
  • non-nucleoside reverse transcriptase inhibitors such as capavirine, delavirdine, efavirenz, etravirine (TMC- 125) and nevirapine;
  • HIV protease inhibitors such as indinivir, nelfinavir, ritonavir, and saquinavir
  • CCR5 antagonists such as TAK-779 or UK-427, 857
  • CXCR4 antagonists such as AMD-3100
  • integrase inhibitors such as L-870,810 or S-1360;
  • investigational drugs such as trizivir, KNI-272, amprenavir, GW-33908, FTC, PMPA, MKC-442, MSC-204, MSH-372, DMP450, PNU-140690, ABT-378, KNI- 764, DPC-083 and/or TMC- 120;
  • antifungal agents such as fluconazole, itraconazole or voriconazole
  • antibacterial agents such as azithromycin
  • the combination therapy includes treatment with a compound as described herein and the "cocktail" therapy described hereinabove. Further according to embodiments of the invention there is provided a use of any of the compounds described herein in the manufacture of a medicament for the treatment of the infections and conditions described herein (e.g., a viral infection caused by a retrovirus).
  • a compound as described herein which is identified for use in the treatment of a viral infection caused by a retrovirus.
  • the compound is utilized in a combination therapy (co-therapy) with at least one agent capable of treating a viral infection caused by a retrovirus.
  • the compounds described herein can be utilized either per se or as a part of a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.
  • a pharmaceutical composition which comprises, as an active ingredient, any of the compounds described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of any of the conditions and infections described herein.
  • the pharmaceutical composition is identified for inhibiting the replication of HIV-I by inhibiting HIV-I RT activity. In some embodiments, the pharmaceutical composition is for the treatment of HIV-I infection.
  • the pharmaceutical composition further comprises an additional agent capable of treating a viral infection caused by a retrovirus, as described herein.
  • a "pharmaceutical composition” refers to a preparation of the compounds presented herein, with other chemical components such as pharmaceutically acceptable and suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • carriers are: propylene glycol, saline, emulsions and mixtures of organic solvents with water, as well as solid (e.g., powdered) and gaseous carriers.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the compounds into preparations which can be used pharmaceutically.
  • Proper formulation is dependent upon the route of administration chosen.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see e.g., Fingl et al., 1975, in
  • the pharmaceutical composition may be formulated for administration in either one or more of routes depending on whether local or systemic treatment or administration is of choice, and on the area to be treated. Administration may be done orally, by inhalation, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, intramuscular or intravenous injection, or topically (including ophtalmically, vaginally, rectally, intranasally).
  • Formulations for topical administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, pills, caplets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.
  • Formulations for parenteral administration may include, but are not limited to, sterile solutions which may also contain buffers, diluents and other suitable additives.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA (the U.S. Food and Drug Administration) approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as, but not limited to a blister pack or a pressurized container (for inhalation).
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.
  • a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising the compound(s) of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of a condition or infection as detailed hereinabove.
  • a Protomol was generated using the default setting of the Surflex program [see, Jain, A. N. Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine. J. Med. Chem. 2003, 46, 499-511].
  • a protomol is an idealized representation of a ligand that makes every potential interaction with the binding site. Using the three dimentional location of the removed inhibitor at the RT binding site in each structure as reference, the Protomol was defined.
  • the Tripos "Leadquest 3" chemical library, containing 46,000 compounds was downloaded from see www.tripos.com/, as a structure data (sd) file.
  • Each compound was then energy- minimized to a single, optimized conformation using the Omega software (Openeye software) and the mmff94s force field and their 3D conformation was saved in a mo 12 file format.
  • a docking process of the compounds onto the RT binding site of Ifk9 and ldtq was then initiated using the generated Protomol.
  • the Docking was performed by fragmenting each molecule and fitting the conformation of each fragment into the Protomol to yield a spatial structure that maximizes molecular similarity to the Protomol.
  • the 10 top conformations for each compound were retrieved according to their score as output files. These included a log output file with the scores for each conformation and a structural output file with the coordinates for the suggested conformations.
  • a chemical library of 46,000 compounds was screened for optimal docking onto the binding site of two of HIV-I RT crystal structures in order to identify novel inhibitors against HIV-I RT.
  • the structures of all screened compounds were energy- minimized and then docked onto the NNRTI binding pocket of the heterodimeric (p66/p51) structure of HIV-I RT, using the Surflex software.
  • Two RT crystal structures were used in the screening process (Ifk9 and ldtq) in order to account for subtle differences between various RT structures, thereby obtaining more reliable docking scores for the screened compounds.
  • the docking conformation of the top ranked structures i.e.
  • RTs Recombinant, wild-type p66/p51 HIV-I RT, derived from BH- 10 clone of HIV-I, was expressed in bacteria. This enzyme, which has six histidines tag at the C-terminus of the p66 subunit, was purified on Ni 2+ nitrilotriacetic acid agarose (Ni-NTA) column followed by cation exchange chromatography, as previously described [Sevilya et al. Nucleic Acids Res 2003, 31, 1481-7].
  • Klenow fragment of E. coli DNA polymerase I (KF), which served as a control, unrelated DNA polymerase was purchased from New England Biolabs.
  • DNA polymerase assay The RNA-dependent DNA polymerase activity of RT was assayed by measuring the incorporation of [ 3 H]dTTP into poly(rA) n .oligo(dT) 1 2 -1 8 template-primer as previously described [Hizi et al. Antimicrob Agents Chemother 1993, 37, 1037-42]. All inhibitors were serially diluted in DMSO and 1 ⁇ l of each concentration (or 1. ⁇ l of DMSO only) was added to 79 ⁇ l of 31.25 mM Tris-HCl pH 7.5, 50 mM KCl and 8 mM MgCl 2 .
  • 10 ⁇ l of purified HIV-I RT 14 ng was added to each tube and the solutions were incubated on ice for 10 minutes.
  • the enzymatic reactions were initiated by the addition of 10 ⁇ l of the substrate mix [50 ⁇ g/ml poly(rA) n «oligo(dT)i 2-18 , 50 ⁇ M dTTP, 150 ⁇ Ci/ml [ 3 HJdTTP] followed by incubation for 30 minutes at 37 °C.
  • the reaction was stopped by adding 50 ⁇ g/ml herring sperm carrier DNA and 40 mM sodium pyrophosphate, followed by precipitation with ice-cold 10 % (w/v) trichloroacetic acid (TCA).
  • the precipitates were collected on Whatman GF/C fiberglass filters, and the filters were washed with 5 % cold TCA and then with 50 % cold ethanol. The dried filters were put in a scintillation fluid and counted in a ⁇ scintillation counter.
  • the DNA-dependent DNA polymerase activity of RT or KF was assayed in a similar manner but with activated herring sperm DNA at the final concentration of 20 ⁇ g/ml, substituting for the synthetic template*primer and with all the four dNTPs present in the reaction mixture, out of which dTTP was 3 H- labeled.
  • the DNA-dependent DNA polymerase activity of RT and of KF was assayed under identical experimental conditions.
  • RNase H assay The RNase H activity of RT was assayed with fluorescence resonance energy transfer technology as previously described [Parniak et al. Anal Biochem. 2003, 322, 33-9]. Briefly, HIV-I RT was incubated with a substrate of hybrid of fluorescein-RNA and DNA- 4-[[40-(dimethylamino)phenyl]azo]benzoic acid and with (or without) a specific inhibitor. Hydrolysis of the substrate and the resulting emitting fluorescence was measured with a fluorescence spectrometer after 30 minutes of incubation at 37 0 C. To improve the signal to noise ratio the assay was slightly modified to contain 6 nM RT and 1 mM DTT. All measurements were done in triplicates in a 96-well plate and IC 5O values were interpolated from the dose response curves.
  • RNA-dependent DNA polymerase activity assays All kinetic assays were conducted as described hereinabove for the quantitative analysis, with different concentrations of either dTTP or poly(rA)n-oligo(dT) I 2-18 template-primer as indicated. Reaction mixtures were incubated for 15 minutes, and the results were linearly fitted using Origin 7.5.
  • Cytotoxicity assays were performed with the XTT substrate as previously described [Roehm et al. J Immunol Methods. 1991, 142, 257-65]. Briefly, B-lymphocytes were incubated in a 96 well plate with various inhibitor concentrations at 37 °C for 72 hours. Cell viability was then assayed with XTT substrate. Absorbance was recorded at 450 nm and reference wavelength was recorded at 620 nm.
  • HIV pseudovirus infection Infectious virions were prepared by transfecting HEK293T cells as previously described [Naldini et al.. Proc. Natl. Acad. ScL U.S.A 1996, 93, 11382-8] with three plasmids: (1) pCMV ⁇ 8.2Gagpol which encodes for HIV- 1 proteins (except Env), (2) pHRCMVGFP, which is transcribed to a mRNA containing both the encapsidation signal and the GFP coding sequences, and (3) pVSV-G, which supplies an envelope protein capacitated to infect wide variety of cells. 25 ⁇ M of Chloroquine was added to the medium prior to transfection.
  • RNA-dependent DNA polymerase activity of HIV-I RT, each at a final 50 ⁇ g/mi concentration (average molar concentration of 156 ⁇ 23 ⁇ M with a range between 105 to 260 ⁇ M).
  • the RNA-dependent DNA polymerase activity (RDDP; i.e. use of RNA as template for transcription of DNA) is unique to RTs and distinct from any other DNA polymerases, which are typically DNA-dependent DNA polymerases (DDDP; which use a DNA as a template), and therefore was tested first.
  • the level of selectivity and specificity of these 71 compounds to HIV-I RT was tested in order to differentiate between specific inhibition and non-specific inhibition of RT due to cross reactivity with unrelated cellular DNA polymerases or interactions with other components of the enzymatic assay. Therefore, the ability of the 71 compounds to inhibit HIV-I RT was compared to its ability to inhibit Klenow fragment of E. coli DNA polymerase I (KF), which served as a distinct and unrelated DNA polymerase. In this case, the ability of the compounds to inhibit DNA-dependent DNA polymerase activity of RT and KF was examined due to KF being a DNA polymerase devoid of any RNA-dependent DNA polymerase activity.
  • KF E. coli DNA polymerase I
  • the selectivity value for only three compounds was between 1 and 1.25 whereas all the other compounds inhibited KF more efficiently than HIV-I RT thereby having a selectivity index below 1.
  • Table 2 below presents the chemical structures, selectivity and activity of the most potent and selective compounds, Compounds 1-8. These eight top molecules were filtered based on a residual RNA-dependnet DNA polymerase (RDDP) activity lower than 15 % and a selectivity index higher than 1.25. Compound 9, despite showing a residual RDDP activity higher than 15 %, was extracted from the tested inhibitors based on its similarity to Compound 7 and tested as all other compounds shown.
  • RDDP RNA-dependnet DNA polymerase
  • the residual activity was calculated by dividing the residual activity, detected in the presence of a final 50 ⁇ g/ml concentration of the tested compound, by the initial activity with no inhibitor present an then multiplying the outcome by 100.
  • Selectivity index was calculates as % residual KF-derived DNA Polymerase (DDDP) activity divided by the % residual HIV-I RT-derived DDDP activity.
  • HIV virions were constructed which carried: (1) viral mRNA backbone into which the coding sequence for the green fluorescent reporter gene was inserted; (2) all HIV associated proteins, which are supplied in trans during packing; and (3) vesicular stomatitis virus (VSV)-G envelope protein, enabling the virions to infect wide range of cells.
  • Productive infection results in an expression of green fluorescent protein (GFP) in the infected cells, which can be monitored with fluorescent microscopy and quantitatively measured by flow cytometry.
  • GFP green fluorescent protein
  • the resulting cells could be separated to uninfected and infected cells, based on their green fluorescent intensity (FIG. IA, right panel).
  • the average fluorescence intensity of the B lymphocytes was then plotted against the concentration of Compound 20 and fitted into a four parameter logistic equation with a corresponding IC 50 value of approximately 168 nM (see, FIG. 1C).
  • FIG. 1C In order to asses whether the quantified fluorescence was due to cells infected by HIV virions, cells subjected to infection with heat-inactivated pseudovirus preparations served as control and as expected did not exhibit any level of fluorescence above background (see, FIG. IA, lowest panel).
  • Compound 20 also exhibited a high selectivity toward
  • HIV-I RT selective cytotoxicity index of 7.4
  • cytotoxicity assays were performed following a 72 hours incubation of the cells with the inhibitor at 37 °C, under the identical experimental conditions used for assaying its HIV transfection inhibitory effect.
  • concentration of inhibitor at which the percent of viable cells was reduced by 50 % was calculated to be about 58 ⁇ M
  • the resulting CC 50 /IC 50 (therapeutic index, TI) value for Compound 20 was calculated to be about 345.
  • Compound 3 for comparison, was less effective than Compound 20 in inhibiting HIV-I pseudovirus infection of B lymphocytes (with an apparent IC 50 value of about 374 nM), but was found to be less cytotoxic, with a calculated therapeutic index above 446 (see, FIG. ID).
  • Nevirapine an NNRTI currently used for HIV-I therapy, was used as a reference and inhibited HIV transfection of B lymphocytes with an IC 50 of 14 ⁇ 3 nM.
  • the inhibition activity of Nevirapine was also tested. Nevirapine exhibited IC 50 values of approximately 1.7 ⁇ M and 0.63 ⁇ M, for the RNA-dependent and DNA-dependent DNA polymerase activities, respectively. As expected, neither compounds (including nevirapine) inhibited significantly the RNase H activity of RT, as similar to most NNRTIs.
  • Yl 81C mutant of HIV-I RT is known to be highly resistant to nevirapine.
  • the Y181C mutant of HIV-I RT was inhibited effectively by compounds 3 and 20 (with apparent IC 50 values of 20 ⁇ M and 70 ⁇ M, respectively), whereby for nevirapine an apparent IC 50 of 100 ⁇ M was observed (see, FIG. 3A).
  • Compounds 3 and 20 also inhibit the activity of HIV-2 RT at relatively high concentrations (e.g., 140 ⁇ M). This feature is unique to these compounds and is not shared by most classic NNRTIs, such as nevirapine.
  • the control calculated value for the reaction without inhibitor was 4.3 ⁇ M dTTP, whereas the calculated Km values in the presence of 0.25, 0.5, 1, and 2 ⁇ M Compound 20 were 4.5, 4.8, 4.3, and 4.2 ⁇ M, respectively (average of 4.5 +0.25 ⁇ M).
  • the kcat values (V max /[RT]) were decreased by Compound 20 from approximately 0.38 s "1 in the absence of inhibitor to 0.28, 0.25, 0.20, and 0.13 s ⁇ ' in the presence of 0.25, 0.5, 1, and 2 ⁇ M Compound 20, respectively.
  • Table 5 presents the chemical structures of all the compounds synthesized to this effect, and the corresponding RT inhibition thereof.
  • the extent of RT inhibition was determined by the extent of reduction in residual HIV-I PV infection in the presence of the inhibitor, at the indicated concentration, according to the procedure described in Example 2 hereinabove.
  • Mass spectra were obtained on a Varian Mat 731 spectrometer.
  • LRMS were obtained on a QToF micro (Waters UK) spectrometer in ESI.
  • a substituted phenylisothiocyanate or a substituted phenylisocyanate the appropriate amine is added and the mixture is stirred at room temperature for the indicated time.
  • an equimolar amount of triethylamine is added. The solvent is thereafter evaporated, and the obtained residue is purified by flash chromatography, so as to obtain the desired compound.
  • Butyric(Z)-N-(3-(butyryloxy)propyl)-N-(4-chloro-2,5-dimethoxyphenyl) carbamimidic thioanhydride (Compound 58): To a solution of Compound 57 (0.19 mmol) in CH 2 Cl 2 (20 ml) at O °C, under N 2 , 4-dimethylamininopyridine (DMAP) (0.19 mmol) and Et 3 N (0.19 mmol) were added. The resulting mixture was stirred for a few minutes and a butyryl chloride (0.19 mmol) in CH 2 Cl 2 (10 ml) was added drop wise. The solution was brought to room temperature and stirred over night.
  • DMAP 4-dimethylamininopyridine
  • Et 3 N (0.19 mmol
  • Compound 59 was obtained by reacting l-chloro-2,5-dimethoxy-4- isothiocyanatobenzene and 3-aminopropyl butyrate and was purified by chromatography (hexane/EtOAc 2:1) to provide the desired Compound 59 as a colorless oil.
  • Ci 4 H 20 N 2 O 4 SCl 37 (MH + , CI + ) 349.0803 found 349.0804 calcd. for Ci 4 H 19 N 2 O 4 SCl 35 (M, CI + ) 346.0754 found 346.077.
  • Butyl 4-(amino-N-(4-chloro-2,5-dimethoxyphenyl)methanethioamino) butanoate (Compound 68): Compound 68 was obtained by reacting l-chloro-2,5- dimethoxy-4-isothiocyanatobenzene and butyl 3-aminobutanoate, was purified by column chromatography (hexane/EtOAc 3:1) and was isolated as a colorless oil in 56 % yield.
  • Compound 69 was obtained by reacting l-chloro-2,5-dimethoxy-4- isothiocyanatobenzene and 4-amino-N-propylbutanamide, was purified by column chromatography (hexane/EtOAc 1:5) and was isolated as a colorless oil in 41 % yield.
  • Compound 70 S-Propyl 4-(amino-N-(4-chloro-2,5-dimethoxyphenyl) methanethioamino) butanethioate (Compound 70): Compound 70 was obtained by reacting l-chloro-2,5- dimethoxy-4-isothiocyanatobenzene and S-propyl 4-aminobutanethioate, was purified by column chromatography (hexane/EtOAc 4: 1) and was isolated as a colorless oil in 10
  • Ethyl 4-(amino-N-(4-chloro-2,5-dimethoxyphenyl)methanethioamino) butanoate (Compound 71): Compound. 71 was obtained by reacting l-chloro-2,5- dimethoxy-4-isothiocyanatobenzene and ethyl 4-aminobutanoate, was purified by column chromatography (hexane/EtOAc 2:1) and was isolated as a yellowish oil in 59 % yield.
  • the compounds described herein bind bonafide the NNRTI-binding hydrophobic pocket of HIV-I RT, which is not found in other DNA polymerases such as the control KF DNA polymerase, used in the selectivity assays conducted. Such compounds should possess some hydrophobic properties to enable binding to this pocket and this feature may also favor their ability to penetrate cells.
  • the docking conformation of these molecules onto the crystal structure of one of the two HIV-I RT used in these studies was visually analyzed.
  • Tyr318 is highly conserved in HIV-I RT and interacts within a 4 A contact distance with most NNRTIs that have been shown to bind the hydrophobic pocket. The importance of a Tyr at position 318 was evident from a mutagenesis analysis, which showed that only when replacing Tyr318 with either Tip or Phe (i.e. Y318F or Y318W RT mutants), the activity of the enzyme substantially retained. While the Y318F mutant still retained a substantial sensitivity to the majority of the NNRTIs tested, the Y318W mutant showed varying degrees of resistance to different NNRTIs.

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Abstract

L'invention concerne des composés qui sont capables d'inhiber une activité d'une transcriptase inverse. Elle concerne en outre des compositions pharmaceutiques contenant ces composés, et des procédés d'inhibition d'une activité de transcriptase inverse et/ou d'un mutant de celle-ci et de traitement d'une infection provoquée par un rétrovirus, au moyen de ces composés. Les composés décrits sont identifiés par le calcul ou sont conçus et nouvellement préparés en se basant sur des caractéristiques structurales identifiées, au moins en partie, par le calcul. Ainsi, l'invention concerne en outre un procédé d'identification de composés candidats pour inhiber une activité de transcriptase inverse de type sauvage et/ou traiter une infection virale provoquée par un rétrovirus.
PCT/IL2008/001555 2007-11-29 2008-11-27 Nouveaux inhibiteurs de transcriptase inverse WO2009069132A2 (fr)

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CN107995910A (zh) * 2015-04-23 2018-05-04 Viiv保健英国第五有限公司 人免疫缺陷病毒复制的抑制剂
CN109761974A (zh) * 2019-01-21 2019-05-17 河南大学 1,2,3,4-四氢-9H-吡啶并[3,4-b]吲哚类TRPV1拮抗剂及其应用
US11613548B2 (en) 2021-02-19 2023-03-28 Sudo Biosciences Limited Substituted pyridines, pyridazines, pyrimidines, and 1,2,4-triazines as TYK2 inhibitors
US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US12084458B2 (en) 2021-02-19 2024-09-10 Sudo Biosciences Limited Substituted pyridines, pyridazines, and pyrimidines as TYK2 inhibitors
US12122785B2 (en) 2023-01-27 2024-10-22 Sudo Biosciences Limited Substituted pyridines, pyridazines, pyrimidines, and 1,2,4-triazines as TYK2 inhibitors

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107995910A (zh) * 2015-04-23 2018-05-04 Viiv保健英国第五有限公司 人免疫缺陷病毒复制的抑制剂
CN109761974A (zh) * 2019-01-21 2019-05-17 河南大学 1,2,3,4-四氢-9H-吡啶并[3,4-b]吲哚类TRPV1拮抗剂及其应用
CN109761974B (zh) * 2019-01-21 2021-05-14 河南大学 1,2,3,4-四氢-9H-吡啶并[3,4-b]吲哚类TRPV1拮抗剂及其应用
US11834441B2 (en) 2019-12-06 2023-12-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US11919887B2 (en) 2019-12-06 2024-03-05 Vertex Pharmaceuticals Incorporated Substituted tetrahydrofurans as modulators of sodium channels
US11613548B2 (en) 2021-02-19 2023-03-28 Sudo Biosciences Limited Substituted pyridines, pyridazines, pyrimidines, and 1,2,4-triazines as TYK2 inhibitors
US12084458B2 (en) 2021-02-19 2024-09-10 Sudo Biosciences Limited Substituted pyridines, pyridazines, and pyrimidines as TYK2 inhibitors
US12103937B2 (en) 2021-02-19 2024-10-01 Sudo Biosciences Limited Substituted pyridines and pyridazines as TYK2 inhibitors
US11827627B2 (en) 2021-06-04 2023-11-28 Vertex Pharmaceuticals Incorporated N-(hydroxyalkyl (hetero)aryl) tetrahydrofuran carboxamides as modulators of sodium channels
US12122785B2 (en) 2023-01-27 2024-10-22 Sudo Biosciences Limited Substituted pyridines, pyridazines, pyrimidines, and 1,2,4-triazines as TYK2 inhibitors

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